Among immuno-compromised cancer patients, organ transplantation patients and AIDS patients, increase of systematic fungal infection or infection by protozoa has become a serious medical problem. The death rate due to such infections is increasing. For these immuno-compromised patients, it is important to treat the fungal infection at an early stage of the infection by administering anti-fungal drugs. The early diagnosis, however, is a difficult task at present. Also many AIDS patients die of Pneumonia by Pneumocystis carinii infection. Recently, it has been reported that β-1,3-glucan exists as a cell wall component of Pneumocystis carinii (Kottom et al., J. Biol. Chem. (2000), 275(51), pp. 40628-34).
To diagnose a fungal infection in immuno-compromised patients, the standard method in mycology, i.e., a method wherein blood is drawn from a patient and cultivated to diagnose the fungal infection has been used. The method has a shortcoming in that the treatment may not be done on time since the result of the diagnosis can be obtained only after 2-5 days. Recently, methods of using the antigens or the metabolites of the fungus have been suggested. However, in the latter case, there is a difficulty in analyzing all of the metabolites, as well as low sensitivity and low accuracy of the diagnosis due to the frequent mutation of the metabolites. For these reasons, research has been widely performed to find a system to recognize accurately the infinitesimal amount of β-1,3-glucan existing in the patient's blood at the early stage of fungal infection.
In the meantime, if the fungal infection occurs during the breeding of lobster, fish or clams, it could cause a severe economic damage to the aquiculture industry since most of the infected fishery may die. Even in this case, if an early diagnosis could be made, the lives of these aquatic animals could be saved if appropriate actions are taken to increase the efficiency of the aquiculture.
Melanin formation in insects is initiated by the oxidation of phenolic compounds existing in the body. Phenoloxidase acting in this process exists in the insect's body as a prophenoloxidase in ordinary times, and the prophenoloxidase is converted to phenoloxidase as the final activated form by the stimulus of the final product of the prophenoloxidase chain reaction. The activation of prophenoloxidase is reported to be initiated by β-1,3-glucan, lipopolysaccharide, peptidoglycan and other cell wall components of the microorganisms.
Prophenoloxidase exists in the body of holometabola and is activated to phenoloxidase by the cascade reaction that is initiated by either β-1,3-glucan or lipopolysaccharide. The reaction system composed of a series of cascade reaction steps is activated easily by foreign pathogens or other molecules from outer system, or inherent factors induced by the degranulation reaction of the insect's own hemocytes. As a result, prophenoloxidase is converted to phenoloxidase to produce melanin by using catecholamines. Therefore, it has been difficult to carry out this reaction system under in vitro conditions.
In U.S. Pat. No. 4,970,152, Ashida and co-workers have suggested a method for determining peptidoglycan or β-1,3-glucan by using a composition comprising a fraction obtained from plasma of silkworm larvae and capable of reacting specifically with β-1,3-glucan or peptidoglycan but not with endotoxin In this patent a composition capable of reacting specifically with β-1,3-glucan has been obtained by removing the substance that reacts with peptidoglycan by affinity chromatography.
Ashida has also reported in Eur. J. Biochem, 188, 507-515(1990) that the divalent ion plays a crucial role in the activation of prophenoloxidase while presenting a composition, separated from mosquito larvae, that recognize β-1,3-glucan.
U.S. Pat. No. 5,266,461 discloses a reagent for determining β-1,3-glucan comprising limulus amebocyte lysate. There is a problem of using this method, however, since limulus is classified as a protected species in most of the countries.